Cell, Vol. 13, 283-271, February 1978, Copyright Q 1978 by MIT

Immunocytochemical Localization of Fibronectin (LETS Protein) on the Surface of L6 Myoblasts: Light and Electron Microscopic Studies

Leo 1. Furcht,*t Deane F. Masher* and Gwen Wendelschafer-Crabb’ *Box 609 Mayo Building Department of Laboratory Medicine and Pathology University of Minnesota Medical School Minneapolis, Minnesota 55455

T St. Paul Regional Red Cross Blood Center St. Paul, Minnesota 55107

$ Department of Medicine University of Wisconsin Medical School Madison, Wisconsin 53706

Summary

Fibronectin (LETS protein) is a major cell surface glycoprotein component of a variety of nontrans- formed, substrate-attached ceils in culture. Its presence has been related to increased adhesive properties. Using the peroxidase-antiperoxidase method to localize antibodies to fibronectin, we have observed that the distribution of fibronectin on L6 myoblasts varies with the density of the culture and the differentiative state of the cells. Low density, undifferentiated cultures of L6 myo- blasts have a sparse accumulation of fibronectin; the antibody-antigen reaction indicates its pres- ence on cell membranes, especially where sev- eral cells are in proximity. Undifferentiated cells in high density cultures have two forms of fibro- nectin localization-a diffuse staining on the mem- brane and a dense staining on an extracellular filamentous matrix. This matrix is composed of filaments ranging from 26-25 nm in diameter which occur singly or coalesce to form bundles. The filaments in this matrix are also observed to have dense globules scattered along their length. These filaments, which are at least in part com- posed of fibronectin, also react with concanavalin A, as do certain plasma membrane components. In contrast to the observations seen in undiffer- entiated cells, differentiated cells or myotubes have a diffuse membrane staining with antifibro- nectin antibodies, and the filamentous form is usually absent.

Introduction

Fibronectin is a glycoprotein composed of high molecular weight subunits (Keski-Oja, Mosher and Vaheri, 1977) and is a major glycoprotein synthe- sized by cultured adherent cells (Ruoslahti et al., 1973; Hynes and Humphreys, 1974; Yamada and Weston, 1974). In cultures, fibronectin is found in conditioned medium, in pericellular structures and

intracellularly (Yamada and Weston, 1974; Mosher et al., 1977b). Transformed, cultured fibroblasts generally lack cell surface fibronectin (Hynes, 1973; Gahmberg, Kiehn and Hakomori, 1974; Vah- eri and Ruoslahti, 1974; for reviews see Hynes, 1976; Vaheri, 1977), which is also known as large external transformation-sensitive (LETS) protein (Hynes and Bye, 1974) and cell surface protein (Yamada and Weston, 1974). When fibronectin is extracted from surfaces of normal cultured fibro- blasts and added to cultures of transformed fibro- blasts, the transformed cells acquire more normal adhesiveness and morphology (Yamada, Ohanian and Pastan, 1976a; Yamada, Yamada and Pastan, 1976b; Ali et al., 1977). Fibronectin binds specifi- cally to collagen (Pearlstein, 1976; Engvall and Ruoslahti, 1977).

In vertebrates, fibronectin is present in the cir- culation [the plasma form is known as cold-insolu- ble globulin (Morrison, Edsall and Miller, 1948; Mosesson and Umfleet, 1970)] and in basal lamina and connective tissue (Linder et al., 1974). When studied by immunofluorescence, fibronectin is found on primitive mesenchymal cells in the devel- oping metanephros and in tubular and glomerular basement membranes of the more mature kidney (Linder, et al., 1974; Wartiovaara, Stenman and Vaheri, 1976). Similarly, fibronectin is found on cells around primative myotomes of the 7 day old chick embryo and later in the sarcolemma sheaths of striated muscle (Linder et al., 1974). Thus major changes in the distribution of fibronectin occur during differentiation.

The L6 rat myoblast line established by Yaffe and Dym (1972) is an excellent cell culture system in which to study myogenesis. In the undifferen- tiated state, cells are well spread and mononucle- ated. As the cells differentiate, they fuse to become multinucleated myotubes, a process associated with increased synthesis of myofibrillar proteins (Patterson and Strohman, 1972), myofilament for- mation (Holtzer et al., 1975), induction of creatine phosphokinase (Shainberg, Yagil and Yaffe, 1971), changes in other enzymes, increases in acetylcho- line receptors (Vogel and Daniels, 1976), and changes in the distribution and mobility of concan- avalin A (ConA) receptors (Furcht, Wendelschafer- Crabb and Woodbridge, 1977). Hynes et al. (1976) iodinated LETS protein (cell surface fibronectin) in cultures of both undifferentiated and differentiated L6 cells. In the present study, we use the unlabeled antibody-enzyme technique (Sternberger et al., 1970) at light microscopic and electron micro- scopic levels to follow changes in distribution of cell surface-associated fibronectin. We find two forms of fibronectin-a filamentous form in extra- cellular spaces and a nonfilamentous form bound

cell 264

diffusely to the plasma membrane. During myo- blast differentiation, the filamentous form of fibro- nectin disappears, but the membrane-bound fibro- nectin is retained. While our experiments were in progress, an immunofluorescence study was re- ported which corroborates the finding that filamen- tous fibronectin is lost during differentiation (Chen, 1977).

Figures la-ld illustrate the typical appearances of heavy metal counterstained sections of L6 myo- blasts as they increase in density and undergo differentiation. Low density myoblasts are mono- nucleated cells with scant extracellular filaments present as seen in Figure la. As the cells approach confluence, undifferentiated L6 cells are often sur- rounded by an extracellular filamentous matrix (EFM) which increases to form a network that

Results literally encircles the myoblasts and fills in the extracellular spaces, as seen in Figure lb. The

Electron Microscopy of L6 Myoblasts filaments comprising the EFM are 26-25 nm in The ultrastructure of L6 myoblasts is revealed by diameter as seen in Figure lc. The filaments occur electron microscopic examination of thin section singly and in bundles, and are frequently seen to of L6 myoblasts cut parallel to the cell monolayer. have electron-dense globules associated with

Figure 1. Electron Micrographs of Heavy Metal-Dounterstained Thin Sections of Myoblasts Depicting Sequential Events of Differentiation and EFM Accumulation

(a) Low density myoblasts are mononucleated fibroblastic type cells. EFM is not present in appreciable amounts (4600x: bar = 1 Am). (b) As proliferation proceeds, a dense extracellular filamentous matrix encircles mononucleated cells in high density cultures, and a point is reached when some cells have formed myotubes. while other populations of cells remain mononucleated (7200x; bar = 1 pm). (c) The filaments which comprise the matrix are 20-25 nm in diameter. Single filaments appear to have electron-dense globules associated with them, and numerous filaments coalesce to form bundles (16.5Wx; bar = 1 pm). (d) Highly differentiated myotubes are multinucleated and have myofilaments organized in more traditional muscle morphology. Very little EFM isassociated with the cell membrane or present in the extracellular space (6600x; bar = 1 pm).

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them. In the same high density cultures, many cells have fused to form multinucleated myotubes. As illustrated in Figure Id, the myotubes are in areas lacking EFM, and their surfaces are relatively free of filamentous material except in areas where undifferentiated mononucleated cells are contact- ing.

Ultrastructural Localization of ConA Ultrastructural localization of ConA on the surface of myoblasts reveals, in Figure 2a, intense staining of the EFM, indicating that the matrix contains exposed mannosyl or glucosyl residues which bind ConA and serve as receptors (Bernhard and Avra- meas, 1974). In Figure 2b, the binding of ConA to myoblast cell surface components is shown to be specific with a-methyl mannoside completely in- hibiting binding of lectin.

lmmunocytochemlcal Localization of Fibronectin Light and electron microscopic localization of fi- bronectin was accomplished on glutaraldehyde- fixed cultures of rat myoblasts using rabbit antihu- man fibronectin (RaFN) and the peroxidase-anti- peroxidase technique. The reaction product ap- pears by light microscopy as dark fibrous areas, and when viewed in color, it can be distinguished from Nomarski shadows.

The precise localization of fibronectin is seen more clearly at the ultrastructural level where we determined that the EFM is reactive to antibody against fibronectin (LETS). In addition to specific staining of EFM, the external plasma membrane of the myoblast also stains. The staining pattern dif-

fers as differentiation progresses. Low density cells in Figure 3 show a light staining pattern on the membrane and a few areas of more dense staining usually at sites of cell contact (these are a cluster of cells in a very low density culture, approximately 30% confluence). Some membranes are completely free of staining. In high density cultures containing mononucleated cells and differentiated myotubes, two patterns of antibody reaction are observed. The contacted mononucleated cells in Figure 4a are surrounded by an EFM which stains heavily for fibronectin, while a diffuse membrane-associated form is also observed. The network of fibronectin encircling the mononucleated cells forms a matrix which appears to be associated with the cell sur- face and the culture dish. This pattern of staining often suggests, as in Figure 4b, that the protein has been secreted by the cell and polymerized in the filamentous form that we observe. At high magnification, in Figure 4c, the antibody staining clearly coats the filaments, and profiles of the peroxidase-antiperoxidase complexes (arrows) are easily discerned. The multinucleated differentiated cells (myotubes), as seen in Figure 4d, are stained in a diffuse manner at the cell membrane, and very little EFM is present. The ultrastructural localiza- tion is representative of what can be visualized as light staining by light microscopy using Nomarski optics (the figure is not enclosed due to the diffi- culty of visualizing in black and white). It should be noted that it is extremely difficult to visualize the diffuse membrane-associated form at the light level.

Controls demonstrate specificity of the staining

Figure 2. Con A Localization

(a) ConA is localized on EFM of high density myoblasts (7600x; bar = 1 pm). (b) ConA localization is performed in the presence of 50 mM a-methyl mannoside (11,000~; bar = 1 Pm).

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Figure 3. Electron Micrograph of the Distribution of Fibronectin Localized with the Unlabeled Antibody Enzyme Technique Low density L6 myoblasts characteristically show a patchy distri- bution of fibronectin. Most staining is seen between areas of cell proximity. Portions of the membrane are lightly stained; others are free of stain (4700x; bar = 1 pm).

reaction for fibronectin at the ultrastructural level. Use of RaFN adsorbed with either bovine globulin or human fibrinogen results in a normal fibronectin staining pattern visualized in Figure 5a. Use of normal rabbit serum (NRS) in the specificity control or RaFN preadsorbed with human fibronectin and fibrinogen results in nonspecific staining of debris located near myotubes; cell membranes and fila- mentous material, however, are free of stain, as seen in Figures 5b and 5c. The nonspecific staining occurs on foamy membranous material which ap- pears to be shed from the membrane. Inasmuch as nonspecific staining of these globular foci is seen in the fibronectin-absorbed controls and with NRS, one can conclude that the staining is not due to antibody to fibronectin. It is probably due to heter- ophile antibody found in both immune and nonim- mune rabbit serum. Use of normal sheep serum (NSS) in place of the linking antibody, goat anti- rabbit gammaglobulin (GaRrG), results in samples which are free of any staining, as in Figure 5d.

Discussion

The current studies have examined the immunocy tochemical localization of fibronectin or LETS pro- tein on the surface of the L6 myoblasts by both light and electron microscopy. We have shown that the EFM and plasma membrane components of L6 myoblasts react with antibody to human fibronectin.

By light and electron microscopy, the majority of fibronectin-specific staining appears as a patchy filamentous form between clusters of cells in low density cultures, and in much larger quantities surrounding mononucleated cells in high density cultures. Our ultrastructural studies show that fi- bronectin occurs in two forms-a dense membrane and substrate-associated EFM, and a diffuse mem- brane-associated nonfilamentous form.

Many immunofluorescent studies have demon- strated fibronectin in fibrils (Wartiovaara et al., 1974; Chen, Gallimore and McDougall, 1976; Chen, 1977; Ali et al., 1977; Yamada, Yamada and Pastan, 1977). Scanning electron microscopy studies of Wartiovaara et al. (1974) showed fibrillar exten- sions from cells which were interpreted to correlate with immunofluorescent localization of fibronectin antibodies. These extensions have also been shown to react with beads coated with antifibro- nectin visualized by scanning electron microscopy (Stenman, Wartiovaara, and Vaheri, 1977). It is difficult to calculate the precise dimensions of the fiber extensions in the work of Wartiovaara et al. (1974), but they do appear to be on the order of 70 nm. These could perhaps correspond to some of the bundles reacting with RaFN which we ob- served.

Furthermore, the studies of Graham et al. (1975) showed the presence of amorphous extracellular material and extracellular fibrils in NIL 8 untrans- formed cells. The diffuse membrane staining seen ultrastructurally is not clearly visualized by either immunofluorescence or Normarski light micros- copy using the peroxidase-antiperoxidase tech- nique. This may result from the relatively sparse distribution of antigenic sites on the plasma mem- brane. The localization of fibronectin, using trans- mission electron mciroscopy with appropriate con- trols, permits greater resolution of antibody bind- ing than is offered by either immunofluorescence or scanning electron microscopy.

Ultrastructural localization of fibronectin in low density cultures reveals a sparse distribution of the glycoprotein between adjacent cells and also coating some of the external membrane surfaces in a diffuse manner. Mononucleated cells in high density cultures demonstrate the two patterns of fibronectin localization. The majority of staining is located on the EFM which is very extensive, and a diffuse membranous staining is apparent on the cell surface. In contrast to these observations seen with undifferentiated cells, fully differentiated my- otubes do not appear to have the dense EFM. Myotubes which lack the EFM, however, do specif- ically bind fibronectin antibodies in a diffuse form to the plasma membrane.

Routine electron microscopic studies with heavy

Fibronectin-lmmunocytochemical Localization, Myoblasts

267

Figure 4. Electron Micrographs of the Distribution of Fibronectin on High Density L6 Myoblasts

(a) High density undifferentiated ceils are surrounded by a dense matrix of EFM which stains specifically for the fibronectin. The membrane is lightly stained (4ooox; bar = 1 pm). (b) Fibronectin is often distributed in high density cultures as “bridges” between nearby cells. The protein appears to be secreted from the cells and occurs in a filamentous form (arrows) which extends beyond the cell’s periphery (6CtWx; bar = 1 pm). (c) At high magnification, the fibronectin localization product is so heavy that it obscures the filaments. Profiles of peroxidase- antiperoxidase complexes are clearly visible (arrows) (14,400x; bar = 1 pm). (d) Multinucleated myotubes have a relatively sparse distribution of fibronectin localization along the cell membrane. Very little EFM is present near differentiated cells (6600x; bar = wm).

metal counterstained thin sections show the devel- opment of the EFM of L6 cells as they increase in density. This matrix is observed to be absent from the fully differentiated myotubes. These filaments appear to be 20-25 nm in diameter and can be rather long, running in bundles that coalesce and split apart. The EFM has binding sites for the lectin ConA indicating exposed mannosyl and glucosyl

residues. Thus it would appear that the matrix has components with antigenic determinants similar or identical to fibronectin and exposed sugar groups reactive with the lectin ConA. Our findings support the observations of Chen (1977) that fibro- nectin in the EFM is lost during differentiation. The diffuse membrane staining is compatible with the studies of Hynes et al. (1976), who showed that

Cell 266

Figure 5. Thin Section Electron Micrographs of Controls for Fibronectin Localization

(a) Mock-adsorbed and bovine globulin-adsorbed RaFN both demonstrate the same distribution pattern which was seen in RaFN-treated monolayers. This micrograph of high density L6 treated with bovine globulin-adsorbed RaFN used as the primary antibody demonstrates this pattern (6600x; bar = 1 am). (b) Human fibronectin-adsorbed RaFN samples are free of specific fibronectin staining; however, the nonspecific circular staining seen in the light micrographs can be seen at the ultrastructural level to be composed of a collection of cell debris (4000x; bar = 1 fim). (c) Specificity control (where NRS is substituted for RaFN) is free of fibronectin-specific staining. Again nonspecific staining is demonstrated as a “bubbly” appearing collection of cell membrane debris which is frequently observed with cell fusion (10,000x; bar = 1

m). (d) Method control (where NSS is substituted for GaRyG) is free of fibronectin-specific staining and nonspecific staining (6600x; bar = 1 crm).

fibronectin could be surface-iodinated in differen- staining in controls using either nonimmune rabbit tiated cultures, and of Chen (1977), who showed serum or immune serum absorbed with fibronectin. binding of iodinated RaFN to differentiated cul- Fibrillar fibronectin on adherent ceils is com- tures. Chen reported that fibronectin was present posed of both disulfide-bonded dimers and multi- in globular foci on differentiated myotubes, and mers (Keski-Oja et al., 1977; Hynes and Destree, we, too, observe such foci by light and electron 1977) and is cross-linked to high molecular weight microscopy. We also, however, visualize such complexes by plasma transglutaminase (Keski-Oja,

Fibronectin-lmmunocytochemical Localization, Myoblasts 269

Mosher and Vaheri, 1976), indicating that pericel- lular fibronectin may become covalently and irre- versibly bound into larger structures, perhaps in the form of the EFM which we observe. Pearlstein (1976) found that urea-solubilized fibronectin me- diates the attachment of BHK 21 to collagen. Born- stein and Ash (1977) demonstrated co-distribution of fibronectin and collagen in the pericellular ma- trix by immunofluorescence.

Our preliminary studies of the EFM in human fibroblast cultures indicate that the EFM is readily removed by trypsin but not by purified bacterial collagenase (Furcht, Mosher and Wendelschafer- Crabb, 1976). Thus the fibrillar structures which we visualized may represent macromolecular com- plexes of fibronectin and other components of the connective tissue matrix; fibronectin, however, ap- pears to be a principal component.

It has been suggested that this class of high molecular weight glycoproteins subserves some role in either cell-cell or cell-substrate adhesive- ness (Yamadaet al., 1976a, 1976b; Ali et al., 1977). It is clear that the presence or absence of fibronec- tin is not pathognomonic of “normal” or trans- formed cells, based on its appearance in undiffer- entiated myoblasts and relative decrease in differ- entiated myotubes. Based on our studies and those of other investigators, (Yamadaet al., 1976b; Pearl- stein, 1976), contacted undifferentiated myoblasts may be more adhesive than fully differentiated myotubes. This seems appropriate, since cells that are fusing to become multinucleated myotubes must undergo some changes where they are less adhesive to the substrate and attach to each other in order to fuse. Associated with myotube forma- tion, there is a decrease in the amount of EFM. This loss of the matrix could be a result of proteo- lytic degradation, but other mechanisms could certainly exist. Fibronectin could be affecting or manifesting changes with differentiation based upon its association with collagen. Collagen and collagen-specific peptides appear to have a role in myogenesis (Hauschka and White, 1972). These collagen peptides block the adherence of a protein similar to fibronectin to collagen-coated dishes (Kleinman, McGoodwin and Klebe, 1976).

The presence of extensive EFM surrounding my- oblasts in high density cultures of undifferentiated cells and the loss of EFM upon differentiation are of special interest based upon other membrane changes that occur. In undifferentiated cells with the EFM, the mobility of ConA receptors is re- stricted, and intramembranous particles seen by freeze fracture are arranged in a clustered distri- bution. Upon differentiation and the loss of the matrix, there is increased mobility of ConA recep- tors, and a uniform distribution of intramembran-

ous particles is observed (Furcht et al., 1977; L. T. Furcht and G. Wendelschafer-Crabb, manuscript submitted). It seems possible that an “exoskele- ton” of the matrix consisting of fibronectin and perhaps other proteins or glycoproteins in an EFM might account for the observations which we have made related to membrane organization and dy- namics. It is conceivable that the matrix, through trans-membranous receptors, could interact with cytoskeletal components and control diverse cel- lular functions.

Exparimental Procedures

Cells and Culture Clonal isolates of LB myoblasts, supplied by Dr. David Schubert (Salk Institute). were plated at 2500/cmz in 35 mm sterile Falcon plates, in Dulbecco’s minimal essential medium supplemented with 10% fetal calf serum [Flow) and grown at 10% CO%, 379: and 10096 humidity, as described (Furcht et al., 1976).

Flbronectln Purltkstlon and Antibody Prepsrstlon Human plasma fibrinogen and fibronectin (cold-insoluble globu- lin) were purified by previously published procedures (Mosher. 1975; Keski-Oja et al., 1977). The purified protein was nearly homogeneous when analyzed on heavily loaded polyacrylamide gels (Figure 6). Antiserum to purified human fibronectin (RaFN) was produced in rabbits by subcutaneous injection of 0.5 mg in complete Freund’s adjuvant followed by repeated subcutaneous injections of antigen (0.2-0.4 mg) at 2-4 week intervals. The antiserum was monospecific when analyzed by double immuno- diffusion against human plasma (Figure 7). The antisera cross- reacted with fibronectin in rat serum and bovine serum (immuno- diffusion plates not shown); absorption with bovine globulin completely removed cross-reacting antibodies to bovine fibronec- tin. Rabbit antiserum prepared in this manner has been shown to immunoprecipitate 58S-methionine-labeled fibronectin specifi- cally from medium and cell extracts of human fibroblast cultures (Mosher, Sakselaand Vaheri, 1977a).

flxatlon for Routlne Electron Mkroscopy For routine electron microscopy, cell monolayers were washed with phosphate-buffered saline without calcium or magnesium (PBS), fixed in 2.5% glutaraldehyde (Electron Microscopy Sci- ences) for 1 hr at 37”c, rinsed with PBS and post-fixed with 1% osmium tetroxide (Electron Microscopy Sciences) in PBS. Sam- ples were then incubated in 1% tannic acid (Mallinckrodt) (Simo- nescu and Simonescu, 1976) in PBS for 15 min, rinsed with 1% sodium sulfate (Baker), dehydrated with ethanol and embedded in situ in Epon (Pelco). Epon embedded monolayers were re- moved from the petri dishes by immersion in liquid nitrogen. Then sections were cut on an LKB Ultratome Ill. The were mounted unsupported on copper grids, stained with lead citrate (Baker) Reynolds, 1963) and viewed with a Philips 300 electron micro- scope operated at 60 kV.

ConA Locallxstlon For ConA localization, cells were prefixed with 1.6% glutaralde- hyde in PBS and then reacted with 50 mg/ml ConA (Miles Yeda, Inc.) for 30 min. Bound ConA was localized by the method of Bernhard and Avrameas (1974). Samples were prepared as de- scribed for routine electron microscopy, except that they were not stained with lead citrate and were viewed at 40 kV.

Immunocytochemkel Locallxatlon of Flbronectln The methods followed procedures described by Sternberger et al. (1970). Cell cultures were fixed in situ with 1% glutaraldehyde

Cell 270

Figure 7. lmmunodiffusion of Adsorbed Rabbit Anti-Human Fi- bronectin

Human plasma was placed in the center well (A), and around the outside were samples of antiserum adsorbed in three different ways: (6) with a mixture of fibrinogen (4 mg/ml) and fibronectin (0.6 mg/m); (C) with fibrinogen (4 mgiml); and (D) with bovine plasma globulins, precipitated with 5W saturated ammonium sulfate and redissolved in 75% of the original plasma volume. The bovine globulin fraction contained approximately 2.5 mg/ml of fibrinogen and 25 mg/ml of other globulins including bovine fibronectin. For adsorption, the antiserum was mixed with equal volumes of the proteins; the mixtures were clotted at 4°C; and the clots and immunoprecipitates were removed by centrifuga- tion

Figure 6. Electrophoresis of Human Plasma Fibronectin (Cold- Insoluble Globulin) in 5% Polyacrylamide Gels Containing Sodium Dodecylsulfate

The conditions of electrophoresis and molecular weight markers have been described previously (Mosher. 1975). The purified protein, 60 wg, was analyzed without (left) and with (right) prior reduction with 1% bmercaptoethanol. Arrows (from top to bot- tom) point to the 4tTO.000 dalton, 200,000 dalton, 65,000 dalton and 46,ooO dalton regions of the gels: The three bands in the 200,000 dalton region in the gel of the nonreduced sample represent partially degraded fibronectin (see Chen, Amramni and Mosesson, 1977). The minor bands of 72,ooO and 60,ooO daltons, which migrated identically in both gels, did not co-migrate with fibrinogen or blood coagulation factor XIII (which were separated from plasma fibronectin in the last steps of the purification), and their identities are not known.

in PBS at 37°C for 30 min. The cells were rinsed with PBS and treated with 0.05 M Tris (Schwartz/Mann)-buffered saline (pH 7.2) plus 1% NSS (Miles) (TBS-NSS) to block nonspecific immu- noglobulin binding. All antibody dilutions were made in TBS- NSS, and all incubations were carried out at room temperature. Samples for fibronectin localization were treated with RaFN at a l/to0 or 11500 dilution for times ranging from 15 min to 24 hr. Staining intensity appeared to be as heavy with l/500 for 15 min as with i/100 for 24 hr. Controls at this stage included a specificity control in which NRS was substituted for RaFN and adsorption controls which include RaFN adsorbed with a mxiture of human fibronectin and fibrinogen, human fibrinogen which should not adsorb antifibronectin antibodies (mock-adsorbed), or bovine globulins (to remove bovine species-specific antifibronectin anti- bodies that would react with fetal calf serum).

Following incubation in the primary antibody, the cells were rinsed with TBS-NSS and then reacted with an excess (l/250) of GaRyG (Cappel) for 10 min. A method control at this step substituted NSS for GaRyG. Cells were again rinsed with TBS- NSS and then reacted with rabbit peroxidase-antiperoxidase com- plex (Cappel) for 10 min. Unreacted peroxidase-antiperoxidase complex was rinsed from the cells with 0.05 M Tris-buffered saline. The peroxidase-antiperoxidase which bound to the RaFN via the GaRyG link was reacted for peroxidase activity. Antibody- treated cells were incubated for 5-10 min in 0.05 M Tris-buffered saline (pH 7.6) containing 12 mg % 3,3’-diaminobenzidine tetra- hydrochloride (Sigma) and 0.0025% H& (Fisher). At this stage, the reaction product for peroxidase was brown in color and could be visualized by light microscopy. Photomicrographs of monolayer cultures in plastic petri dishes were taken with an Olympus BH microscope with Nomarski optics and an OM-10 automatic camera system. To render the reaction product elec- tron-dense for ultrastructural studies, reacted cells were treated with 1% osmium tetroxide in PBS. Cells were then routinely prepared in situ for electron microscopy. Sections, however, were viewed without heavy metal counterstain and at 40 kV to enhance contrast of the reaction product.

Acknowledgments

We wish to thank S. Gentry, R. Scott, J. lrke and S. Palm for technical assistance, and C. Furcht for assistance in the prepara- tion of this manuscript. This work was supported by a Basil O’Connor starter research grant from the National Foundation March of Dimes, the NIH and the Leukemia Task Force to L.T.F. and the University of Wisconsin Graduate School, and an institu- tional grant to the University of Wisconsin from the American Cancer Society to D.F.M.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be

Fibronectin-lmmunocytochemical Localization, Myoblasts 271

hereby marked “adverfisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received October 20.1977; revised November 28,1977

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Note Added In Proof

Hedman, Wartiovaara and Vaheri (J. Cell Biol.. in press) have found a somewhat similar ultrastructural localization of fibro- nectin on human fibroblasts.